王 娟，楊葉寧，秦 晶，唐玉嫻，李清葉，蘇 琦，譚 暉

(1.南華大學(xué)腫瘤研究所，湖南 衡陽(yáng) 421001，2. 解放軍總醫(yī)院呼吸科，北京 100853)

二烯丙基二硫下調(diào)DJ-1抑制人白血病HL-60細(xì)胞增殖及誘導(dǎo)分化

王 娟1，2，楊葉寧1，秦 晶1，唐玉嫻1，李清葉1，蘇 琦1，譚 暉1

(1.南華大學(xué)腫瘤研究所，湖南 衡陽(yáng) 421001，2. 解放軍總醫(yī)院呼吸科，北京 100853)

目的 研究二烯丙基二硫(DADS)通過下調(diào)DJ-1對(duì)人白血病細(xì)胞系HL-60增殖抑制及誘導(dǎo)分化的影響。 方法 通過細(xì)胞形態(tài)法觀察細(xì)胞分化；Western blot及免疫細(xì)胞化學(xué)檢測(cè)DADS對(duì)HL-60細(xì)胞DJ-1表達(dá)的影響；針對(duì)DJ-1 mRNA序列設(shè)計(jì)合成siRNA轉(zhuǎn)染人白血病細(xì)胞HL-60，Western blot檢測(cè)基因干擾效果；利用MTT法及間接免疫熒光檢測(cè)DADS及DJ-1沉默對(duì)HL-60細(xì)胞的生長(zhǎng)及細(xì)胞分化的影響。結(jié)果 DADS可以誘導(dǎo)HL-60細(xì)胞向成熟粒細(xì)胞系樣分化。DADS呈時(shí)間依賴性抑制DJ-1的表達(dá)(P<0.05)，DJ-1干擾組細(xì)胞生長(zhǎng)減慢(P<0.05)，與DADS 共同作用后可誘導(dǎo)HL-60細(xì)胞分化。 檢測(cè)細(xì)胞生長(zhǎng)情況發(fā)現(xiàn)干擾組細(xì)胞生長(zhǎng)減慢(P<0.05)。結(jié)論 DADS通過下調(diào)DJ-1抑制細(xì)胞增殖并誘導(dǎo)HL-60細(xì)胞分化;干擾DJ-1基因可以增強(qiáng)DADS對(duì)HL-60細(xì)胞的增殖抑制誘導(dǎo)分化作用。

白血??；DJ-1；DADS；誘導(dǎo)分化；基因沉默；增殖

白血病是常見惡性腫瘤之一，且各種治療效果差。隨著誘導(dǎo)分化和誘導(dǎo)凋亡藥物出現(xiàn)，白血病的治療發(fā)生了突破性的進(jìn)展。因此，開發(fā)高效、低毒治療白血病的藥物，尋找有效靶點(diǎn)進(jìn)行靶向干預(yù)治療，提高5年生存率，對(duì)白血病治療具有重要的意義。

大蒜及其烯丙基硫化物在腫瘤防治方面的作用研究受到國(guó)內(nèi)外學(xué)者的廣泛關(guān)注。二烯丙基二硫(diallyl disulfide, DADS)對(duì)胃癌、食管癌、乳腺癌、肺癌、前列腺癌、結(jié)腸癌、等腫瘤均有明顯的抑制作用[1-5]。

近年來研究發(fā)現(xiàn)，DJ-1 高表達(dá)于肺癌、食道癌、胰腺癌、肝癌、乳腺癌、喉癌、黑色素瘤等惡性腫瘤中[6-8]，DJ-1表達(dá)與腫瘤的T分期、臨床分期、淋巴結(jié)轉(zhuǎn)移、腫瘤分化、腫瘤的復(fù)發(fā)呈相關(guān)性。另外，上調(diào)DJ-1基因的表達(dá)可促進(jìn)細(xì)胞的癌變，降低化療藥物對(duì)癌細(xì)胞的增殖抑制作用，與化療抵抗有關(guān)[9-10]。這些研究表明，DJ-1 促癌基因可用于診斷和預(yù)測(cè)惡性腫瘤病人的預(yù)后，具有潛在的臨床應(yīng)用價(jià)值。因此，DJ-1蛋白可作為候選的腫瘤標(biāo)志蛋白，也可作為抗腫瘤藥物作用的重要靶點(diǎn)。

本研究通過熒光標(biāo)記siRNA沉默HL-60細(xì)胞內(nèi)DJ-1基因表達(dá)，觀察沉默DJ-1以及DADS作用下對(duì)HL-60細(xì)胞生物學(xué)影響，證實(shí)DJ-1可能是DADS抑制HL-60細(xì)胞增殖及誘導(dǎo)分化的靶點(diǎn)，擬闡明DADS抗白血病的分子機(jī)制，從而為開發(fā)高效、低毒的抗白血病藥物與基因干預(yù)治療提供理論基礎(chǔ)和實(shí)踐依據(jù)。

1 材料與方法

1.1 材料人早幼粒白血病HL-60細(xì)胞系，中南大學(xué)細(xì)胞中心饋贈(zèng)；DADS為Fluka公司產(chǎn)品；小牛血清，杭州四季青生物工程公司產(chǎn)品；MTT：Amreco公司產(chǎn)品；DJ-鼠抗人單克隆抗體，購(gòu)自Milipore公司；β-actin，鼠抗人單克隆抗體，聯(lián)科生物公司；CD11b兔多克隆抗體，購(gòu)自Epitomics公司，Cy3標(biāo)記的山羊抗兔二抗購(gòu)自康為世紀(jì)生物科技有限公司；Western blot灌膠試劑盒均購(gòu)自北京康為世紀(jì)生物公司；siRNA片段，購(gòu)自廣州銳博生物技術(shù)公司。

1.2 方法

1.2.1 體外細(xì)胞培養(yǎng)及細(xì)胞形態(tài)學(xué)觀察 HL-60細(xì)胞懸浮培養(yǎng)于含體積分?jǐn)?shù)為0.12的熱滅活小牛血清的RPMI 1640培養(yǎng)基中，在37℃、含體積分?jǐn)?shù)為0.05的CO2培養(yǎng)箱內(nèi)培養(yǎng)。將對(duì)照組和經(jīng)1.25 mg·L-1DADS處理后4 d的HL-60細(xì)胞， Giemsa染色，顯微鏡鏡下觀察細(xì)胞形態(tài)變化，拍照。

1.2.2 Western blot驗(yàn)證DJ-1蛋白的表達(dá) 離心收集細(xì)胞；提取細(xì)胞總蛋白，BCA蛋白定量。蛋白變性后根據(jù)蛋白濃度加入適量蛋白，再進(jìn)行SDS-PAGE凝膠電泳；用蛋白濕轉(zhuǎn)移裝置將蛋白轉(zhuǎn)移到PVDF膜上；5%脫脂牛奶封閉液；加入稀釋的Ⅰ抗；再加入稀釋的Ⅱ抗室溫孵育1 h；顯影。

1.2.3 細(xì)胞免疫化學(xué)法觀察DADS對(duì)HL-60細(xì)胞DJ-1的影響 離心收集細(xì)胞，涂片；體積分?jǐn)?shù)為0.95的乙醇固定10 min，每張切片滴加50 μL即用型血清封閉液，室溫孵育10 min后甩干；滴加一抗工作液50 μL，4 ℃孵育過夜；每張切片加50 μL生物素標(biāo)記的二抗，室溫孵育30 min；每張切片加50 μL鏈霉素抗生物素蛋白-過氧化酶溶液，室溫孵育30 min；每張切片加100 μL新鮮配制的DAB溶液，顯色3～5 min；蘇木精復(fù)染，酒精脫水，中性樹膠封片。

1.2.4 細(xì)胞瞬時(shí)轉(zhuǎn)染及篩選有效的DJ-1 siRNA序列 取對(duì)數(shù)生長(zhǎng)期HL-60細(xì)胞接種至24孔板，每孔加入不含抗生素的培養(yǎng)基進(jìn)行轉(zhuǎn)染。用不含血清的DMEM分別稀釋Lipo2000和siRNA，室溫孵育5 min；最后形成siRNA-Lipo2000混和物室溫孵育20 min。將siRNA-Lipo2000混和液加入每個(gè)含有細(xì)胞及培養(yǎng)液的孔中。將培養(yǎng)板放入培養(yǎng)箱中孵育4～6 h后，熒光顯微鏡下觀察拍照，繼續(xù)培養(yǎng)。轉(zhuǎn)染48和72 h后,提取總蛋白，進(jìn)行Western blot檢測(cè)。DJ-1 siRNA序列取序列a、b、c中抑制率最高組。

1.2.5 MTT法檢測(cè)細(xì)胞生長(zhǎng) 離心收集對(duì)數(shù)生長(zhǎng)期HL-60細(xì)胞，制備細(xì)胞懸液接種至96孔板，邊緣孔用無菌PBS填充，每板設(shè)對(duì)照組，加培養(yǎng)基100 μL，每組設(shè)定6個(gè)復(fù)孔；置入培養(yǎng)箱孵育48 h；每孔加入10 μL MTT溶液，繼續(xù)孵育4 h；取出培養(yǎng)板后離心，每孔加入100 μL二甲基亞砜，置搖床上低速震蕩20 min，使結(jié)晶物充分溶解后，酶聯(lián)免疫檢測(cè)儀測(cè)量各孔吸光度值。

1.2.6 間接免疫熒光細(xì)胞化學(xué)實(shí)驗(yàn) 離心收取對(duì)數(shù)生長(zhǎng)的細(xì)胞，PBS洗滌細(xì)胞，取10 μL細(xì)胞懸液滴置載玻片，室溫自然干燥，滴加50 μL丙酮固定約5min，羊血清37℃孵育30min，滴加稀釋后的CD11b抗體，37℃孵育1 h，孵育二抗：滴加稀釋后的熒光標(biāo)記二抗，室溫、避光孵育40 min，熒光顯微鏡下觀察涂片，拍照。

2 結(jié)果

2.1 DADS處理HL-60細(xì)胞前后形態(tài)學(xué)變化姬姆薩染色結(jié)果顯示，DADS處理HL-60細(xì)胞96 h后，細(xì)胞形態(tài)發(fā)生改變，主要表現(xiàn)在胞核的分化，呈現(xiàn)U型或腎型改變，核質(zhì)比例減少，核仁減少或消失等。對(duì)照組則細(xì)胞體積較大，胞核大且圓，見Fig 1。表明DADS有誘導(dǎo)HL-60細(xì)胞分化的作用。

Fig 1 Detection of cell differentiation by Giemsa stain(×400)

A:HL- 60 cells B:HL- 60 cells treated by DADS for 96h

2.2 DADS抑制HL-60細(xì)胞DJ-1蛋白的表達(dá)Western blot結(jié)果顯示，與對(duì)照組比較，DADS作用HL-60細(xì)胞24、48、72及96 h后，DJ-1蛋白表達(dá)呈時(shí)間依賴性明顯下降(Fig 2)。這提示DADS可以抑制DJ-1蛋白的表達(dá)。

Fig 2 The effect of DADS on the expression of DJ-1 in HL-60 cells detected by Western blots

1:Control group;2: Treated by DADS for 24h;3: Treated by DADS for 48h;4: Treated by DADS for 72h;5: Treated by DADS for 96 h.*P<0.05,**P<0.01vscontrol

2.3 免疫細(xì)胞化學(xué)觀察DADS對(duì)HL-60細(xì)胞DJ-1蛋白表達(dá)的影響DJ-1在HL-60細(xì)胞中呈強(qiáng)陽(yáng)性表達(dá)，而 DADS處理72 h 后， DJ-1表達(dá)明顯下降，見Fig 3，這進(jìn)一步表明DADS可以抑制DJ-1蛋白的表達(dá)。

Fig 3 The effect of DADS on the expression of DJ-1 in HL-60 cells detected by immunocytochemistry

A: HL-60 cells; B: HL-60 cells treated by DADS for 72h

2.4 Western blot檢測(cè)轉(zhuǎn)染前后DJ-1蛋白表達(dá)以及DADS對(duì)其作用的效果將Cy3熒光標(biāo)記的DJ-1-siRNA瞬時(shí)轉(zhuǎn)染HL-60細(xì)胞后，6 h后熒光顯微鏡下觀察細(xì)胞內(nèi)見紅色熒光標(biāo)記物表達(dá)，轉(zhuǎn)染效率達(dá)95%以上，提示轉(zhuǎn)染成功。干擾組DJ-1蛋白表達(dá)較對(duì)照組、脂質(zhì)體組及陰性對(duì)照組明顯下調(diào)(P<0.05)，見Fig 4；DADS分別處理對(duì)照組、干擾組、脂質(zhì)體組及陰性對(duì)照組后，DJ-1蛋白表達(dá)較未處理前下調(diào)，且DADS處理干擾組前后DJ-1蛋白表達(dá)差異有顯著性(P<0.05)，見Fig 5。

Fig 4 The effect on the expression of DJ-1 in HL-60 cells treated by siRNA as detected by Western blots

1:Control group;2:Negative control group;3:Liposome group;4:DJ-1 siRNA group.*P<0.05vscontrol

2.5 MTT檢測(cè)轉(zhuǎn)染前后細(xì)胞增殖的影響MTT法證實(shí)，干擾組細(xì)胞生長(zhǎng)較對(duì)照組、脂質(zhì)體組及陰性對(duì)照組減慢，差異有顯著性意義(P<0.05)，見Fig 6。這表明干擾DJ-1可以抑制HL-60細(xì)胞的增殖能力。

2.6 間接免疫熒光檢測(cè)DJ-1干擾及DADS對(duì)HL-60細(xì)胞分化的影響CD11b是成熟髓細(xì)胞表面分化抗原，DADS作用前，對(duì)照組細(xì)胞及脂質(zhì)體組幾乎不表達(dá)標(biāo)記紅色熒光的CD11b、DJ-1干擾組CD11b表達(dá)增高，DADS作用后，3組細(xì)胞CD11b的表達(dá)明顯提高，提示DADS具有誘導(dǎo)HL-60細(xì)胞分化的作用，DJ-1干擾可以促進(jìn)DADS誘導(dǎo)的HL-60細(xì)胞分化，見Fig 7。

Fig 5 The expression of DJ-1 in HL-60 cells treated by DADS before and after transfection

1:Control group treated by DADS;2:Negative control group treated by DADS;3:DJ-1 siRNA group treated by DADS;4:Liposome treated by DADS.*P<0.05vscontrol

Fig 6 The effect of DJ-1 on the proliferation of HL-60 cells detected by MTT

*P<0.05vscontrol

3 討論

Fig 7 The effect on the expression of CD11b in HL-60 cells detected by indirect immunofluorescence assay

A:HL-60 cells;B:Liposome group;C:DJ-1 siRNA group;1:DADS(-)2:treated by DADS for 72h

DJ-1編碼蛋白屬于ThiJ/PfpI/DJ-1超家族，是具有多種功能的蛋白質(zhì)。DJ-1在多種惡性腫瘤中特異性表達(dá)， 而未見于正常組織。有研究發(fā)現(xiàn)， RNAi沉默DJ-1在白血病細(xì)胞系K562以及HL-60的表達(dá)，抑制了細(xì)胞增殖且增加了白血病細(xì)胞對(duì)化療藥物依托泊苷的敏感性。用特異性DJ-1-siRNA干擾神經(jīng)母細(xì)胞瘤細(xì)胞株SH-SY5Y細(xì)胞后暴露于百草枯，能增強(qiáng)SH-SY5Y細(xì)胞對(duì)百草枯引起凋亡的敏感性。因此可以認(rèn)為，DJ-1可作為多種惡性腫瘤治療的一個(gè)潛在靶點(diǎn)。有人采用蛋白質(zhì)組學(xué)研究發(fā)現(xiàn)，DJ-1蛋白高表達(dá)與維甲酸、順鉑等藥物耐藥有關(guān)[11-12]。

先前已證實(shí)小劑量DADS主要使HL-60細(xì)胞阻滯于G1期，S期細(xì)胞減少，DNA合成受阻， 細(xì)胞不能進(jìn)入分裂周期而轉(zhuǎn)入靜息狀態(tài)，在藥物的刺激下誘導(dǎo)其向正常細(xì)胞分化[13]。運(yùn)用蛋白質(zhì)組學(xué)初步鑒定DADS誘導(dǎo)人白血病HL-60細(xì)胞的差異蛋白質(zhì)18 種，其中DJ-1表達(dá)下調(diào)[14-15]，利用Western blot進(jìn)行驗(yàn)證，表明DADS可以通過降低DJ-1的表達(dá)誘導(dǎo)HL-60細(xì)胞分化，DJ-1siRNA轉(zhuǎn)染人白血病HL-60細(xì)胞后，可抑制細(xì)胞增殖，并且隨著DADS作用時(shí)間的延長(zhǎng)，對(duì)細(xì)胞增殖的抑制作用愈明顯。

DJ-1促進(jìn)癌細(xì)胞分裂增殖和遷移侵襲，很可能涉及到幾個(gè)相互協(xié)調(diào)的細(xì)胞內(nèi)分子途徑。研究證實(shí)，DJ-1蛋白是PTEN 抑癌基因的主要負(fù)性調(diào)控蛋白之一, PTEN拮抗著調(diào)節(jié)細(xì)胞生長(zhǎng)和生存的PI3激酶和PKB/Akt信號(hào)通路,在癌癥患者血漿中發(fā)現(xiàn)DJ-1的含量和表達(dá)量增加，刺激PI3K/Akt通路促進(jìn)腫瘤細(xì)胞的增殖與生長(zhǎng)[16-17]。同時(shí)DJ-1作為一種抑制PTEN的蛋白，影響細(xì)胞的黏附、運(yùn)動(dòng)與遷移。FAK信號(hào)通路與惡性腫瘤細(xì)胞的侵襲和轉(zhuǎn)移有著密切的關(guān)系，有實(shí)驗(yàn)證實(shí)干擾PTEN后，可以激活Fak通路，參與肝癌、胃癌、白血病細(xì)胞遷移及侵襲[18-19]。因此考慮DJ-1對(duì)部分信號(hào)通路的過度激活起著舉足輕重的作用。

綜上所述，DJ-1可能通過調(diào)控細(xì)胞分裂、增殖、遷移、分化以及凋亡，參與白血病的發(fā)生、發(fā)展以及預(yù)后，可能是白血病一個(gè)潛在的治療靶點(diǎn)，亦或是白血病的一個(gè)潛在生物標(biāo)志物，同時(shí)預(yù)示著DADS可能成為臨床白血病治療的有效藥物。

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Diallyl Disulfide down regulated DJ-1 inhibiting ability of proliferation and inducing human leukemic cell differentiation

WANG Juan1,2,YANG Ye-ning1,QIN Jing1,TANG Yu-xian1,LI Qing-ye1,SU Qi,TAN Hui1

(1.CancerResearchInstitute,NanhuaUniversity,HengyangHunan421001,China;2.RespiratoryofChinesePLAGeneralHospital,Beijing100853,China)

Aim To observe the effect of DADS on the poliferation and differentiation of human leukemic cells by down-regulating DJ-1.Methods Cell differentiation were detected by morphologic observation.The effects of DADS on DJ-1 expression in HL-60 cells were detected by immunocytochemistry and Western blot. DJ-1-specific siRNA was designed and synthesized. Small interfering RNA was transfected into HL-60 cells to knock down the DJ-1 expression.Western blot was used to detect the siRNA interference efficiency.The effects of DADS and DJ-1 silence on cell growth and differentiation in HL-60 cells were detected by MTT method and indirect immunofluorescence detection.Results HL-60 cells were induced differentiation toward neutrophilic by DADS.The expression of DJ-1 was inhibited in HL-60 cells after treated with DADS in a time dependent manner(P<0.05).DJ-1 protein was overexpressed in HL-60 cell lines, which could be reversed after treated with DADS.Fluorescently labeled DJ-1-siRNA transfected HL-60 cells. All these detections showed that the growth rate of DJ-1-siRNA group was lower(P<0.05).Conclusion DADS inhibits cell proliferation and induces cell differentiation by down-regulating DJ-1 in HL-60 cell lines.Silencing DJ-1gene enhances the rate of inhibition of proliferation and induction differentiation of HL-60 cell lines after treated with DADS.

leukemia；DJ-1；DADS； inducing differentiation；gene silence；proliferation

時(shí)間：2015-3-3 11:08 網(wǎng)絡(luò)出版地址：http://www.cnki.net/kcms/detail/34.1086.R.20150303.1108.024.html

2014-11-22,

2014-12-26

國(guó)家自然科學(xué)基金資助項(xiàng)目(No 81100375)；湖南省高校創(chuàng)新平臺(tái)開放基金(No 11K057)；南華大學(xué)博士科研啟動(dòng)基金 (No 2010XQD40)

王 娟(1983-)，女，碩士生，研究方向：白血病防治的分子機(jī)制，E-mail：630811881@qq.com； 楊葉寧(1988-)女，碩士生，研究方向：白血病防治的分子機(jī)制,共同第一作者，E-mail：27199139@qq.com; 譚 暉(1973-)，女，博士，碩士生導(dǎo)師，研究方向：白血病防治的分子機(jī)制，通訊作者，F(xiàn)ax：0734-8281547，E-mail：1047550723@qq.com

10.3969/j.issn.1001-1978.2015.03.024

A

1001-1978(2015)03-0416-05

R329.24；R394.2；R733.702.2；R916.4